Over the last three decades, amplification techniques such as polymerase chain reaction (PCR), ligase chain reaction (LCR) or strand displacement amplification (SDA) tests, have become important, if not even essential, tools in research and clinical applications. As such, PCR is the most commonly used method to amplify specific nucleic acid species. The amplified nucleic acid sequences can be effectively detected, quantified, and analysed either directly in the amplification reactions or in subsequent downstream applications, such as sequencing or electrophoresis. Due to its high specificity, efficiency, and sensitivity, amplification-based techniques are applied widely in the fields of basic biological research, biomedical research, applied testing, and molecular diagnostics.
For example, many massively parallel sequencing (or “next generation sequencing”, NGS) platforms—while differing in engineering configurations and sequencing chemistry—share the technical paradigm of massive parallel sequencing via spatially separated, clonally amplified DNA templates or single DNA molecules in a flow cell. In order to prepare sequencing templates, nucleic acid fragments are first ligated with platform-specific sequencing adaptors to generate sequencing libraries, which step is generally followed by a PCR step in order to achieve sufficient amount of library molecules that can be used in the next steps. Further, in a second step, DNA sequencing templates are generated by clonal amplification of the sequencing library molecules in vitro to generate thousands to hundreds of thousands of the identical copies from the same sequencing library molecule.
Due to their high sensitivity, amplification techniques are prone to contamination giving false or inaccurate results, since the repeated amplification of the same target sequence leads to accumulation of amplification products in the laboratory environment, where also plasmid clones derived from organisms that have been previously analysed may be present in large numbers. One of the major risks posted by the PCR-based nucleic acid analysis is carry-over contamination, where the contamination of the PCR reaction with residual products from previous rounds of the PCR can lead to false or inaccurate positive results.
Also, e.g. with respect to NGS, it is a known fact that appliances requiring DNA amplification may introduce sequencing errors, since PCR can introduce errors in the amplified templates. In particular AT-rich and GC-rich target sequences often show amplification bias, which results in their underrepresentation in genome alignments and assemblies.
Thus, in order to avoid biased amplification reactions, amplicon carryover contamination of reaction tubes with previously generated amplicons needs to be prevented, and/or previously generated amplicons need to be “inactivated” or destroyed, such, that they are ineligible targets for downstream amplification reactions.
Several attempts to minimize the occurrence of errors during PCR have been made and different techniques prior and after the amplification reactions have been developed.
One strategy to prevent carry-over contaminants when amplifying DNA and RNA is to have a separate lab for set-up and amplification, the minimization of the number of pipetting steps, and the prevention of the opening of the tube after amplification. However, apart from the practical impediments, this method offers no guarantee for avoiding carry-over contaminations.
Another strategy to prevent carry-over contamination is the dUTP/UNG method, where dTTP is partially or completely replaced by dUTP during PCR amplification, thereby producing amplicons containing deoxyuracil (dU); subsequent PCR mixtures are then pretreated with uracil-N-glcyosylase (UNG), an enzyme recognizing and removing uracil residues. If a dU-containing contaminant from a previous PCR is present in a new, subsequent PCR, it will be cleaved by a combination of UNG digestion and the high temperature of the initial denaturation step of the subsequent PCR; after treatment, such contaminants cannot serve as a PCR template, since any newly added DNA template contains thymidine instead of uridine, and is, thus, not affected by this procedure.
However, the dUTP/UNG method cannot be used to prevent carry-over contamination in high-fidelity PCR with proofreading polymerases, which are frequently used to ensure sequencing accuracy: The activity of those proof-reading polymerases is normally inhibited by the presence of the dUTP in the reaction mix. Moreover, the proof-reading polymerases are normally stalled once encountering uracil bases on the DNA template and polymerization cannot continue beyond the uracil bases.
Another measure is the method of primer hydrolysis using uniquely synthesized chimeric primers which can be removed from the PCR products after cleavage of the latter, thus generating truncated amplicons lacking primer binding sites. Thus, this amplicons will not be recognized as targets in subsequent amplification reactions. However, also this method provides opportunities for contamination and, above all, primer hydrolysis protocol strongly vary in their efficiency.
Thus, despite the improvements and the methods established over the last decades, there still is the need to effectively avoid carry-over contaminations, and to thus minimize the risk of false, incorrect or false positive results.
Against this background, it is an object of the present invention to provide for novel methods to improve amplification processes in general, specifically to minimize the risk of carry-over contamination of amplification products.
The present invention satisfies these and other needs.